A. Field of the Invention
The invention relates to a method and apparatus for preparation of samples that are to be subjected to mass spectrometry analysis. Specifically, the invention relates to methods for preparing samples for mass spectrometry analysis and to plate-like members used to position samples on a sample plate for subsequent mass spectrometry analysis.
B. Background Information
Mass spectrometry devices measure the molecular mass of a molecule by measuring the molecule""s flight path through a set of magnetic and electric fields. Such devices are well known and are widely used in the field of bio-molecular research. In proteomics research, for instance, mass spectrometry is used to identify proteins.
Proteins are typically separated from one another by electrophoresis, such as the techniques described and claimed U.S. Pat. No. 5,993,627 to Anderson et. al. (the Anderson et. al. patent), which is incorporated herein by reference in its entirety. For instance, as set forth in the Anderson patent, a tissue sample is first subjected to a first dimension electrophoresis process where groups of proteins are separated linearly within a tubular gel filled column. The first dimension separation of proteins is then inserted along an edge of a flat planar gel slab and subjected to a second dimension of electrophoresis thereby generating a two dimensional pattern of spots formed by clusters of proteins that have moved to respective iso-electric focusing points. Thereafter, selected proteins are excised from the second dimension gel slab for further study. The selected excised spots are next prepared for analysis using, for instance, mass spectrometry.
An increasingly used technique for studying biological molecules includes the use of MALDI mass spectrometry apparatus (matrix-assisted laser desorption ionization apparatus) where the biological sample is embedded in a volatile matrix and is vaporized by being subjected to an intense laser emission. One such MALDI apparatus is a MALDI-TOF apparatus (TOF is time-of-flight spectrometry). In the field of proteomics, mass spectrometry, and in particular, MALDI-TOF techniques are used to determine the molecular weight of peptides produced by digestion of isolated proteins. One such MALDI-TOF apparatus is VOYAGER DE STR Biospectrometry Workstation manufactured and sold by APPLIED BIOSYSTEMS.
FIG. 1 depicts a generic MALDI-TOF apparatus that includes a frame 1 that supports the electronic and computer equipment necessary to control a laser 5. The laser 5 is aimed at a fixed location in a positioning mechanism 10. The positioning mechanism 10 includes means (not shown) for positioning a sample in the line of fire of the laser 5. Typically in a MALDI-TOF apparatus, the laser is fixed in place and the sample is moved into position for analysis. The depicted MALDI-TOF apparatus includes a small removable sample plate 15, shown in FIG. 2, that fits into the positioning mechanism 10. Typically, the sample plate 15 is insertable into a slot 20 in the positioning mechanism 10 of the MALDI-TOF apparatus and is thereafter held in a specific orientation within the positioning mechanism 10 for sample analysis. The sample plate 15 typically holds a plurality of discrete samples on one surface thereof, with the samples being spaced apart from one another. The sample plate 15 includes guide members 15a, guide holes 15b and alignment pin 15d that are used by corresponding members (not shown) within the positioning mechanism 10 for positioning and moving the sample plate 15 with respect to the line of fire of the laser 5.
The samples are typically loaded onto the sample plate 15 by a separate device or robotic apparatus, such as the X-Y robotic apparatus depicted in FIG. 3. Such X-Y robotic apparatuses are typically manufactured and sold with each specific mass spectrometry apparatus. The X-Y robotic apparatus depicted in FIG. 3 includes a recess that retains the sample plate 15 in position for sample loading. The X-Y robotic apparatus includes a first arm 30 that moves back and forth along an X axis and a second arm 40 that moves along a Y axis defined along the length of the first arm. The second arm 40 supports a pipette tip 45 that is used to spot samples on the sample plate 15. Specifically, the pipette tip 45 is moved by the first and second arms 30 and 40 to a position above a 96-well plate 50 (or other similar sample holder) or microcentrifuge tubes and the pipette tip 45 picks up a sample from the 96-well plate 50 or microcentrifuge tubes. The pipette tip 45 is then moved to a location above the sample plate 15 and the sample is spotted on the specified location of the sample plate.
Typically, an array of samples are spotted on the sample plate 15 at predetermined locations. After the array of samples are loaded onto the sample plate 15, the sample plate 15 is inserted into the slot 20 of the MALDI apparatus. Using an imaging system 25 focused on the sample plate 15 within the MALDI apparatus, in combination with the positioning mechanism 10, the laser beam from the laser 5 can be aimed, one by one, at the sample(s) on the sample plate 15.
The mass spectrometry apparatus typically takes several hours to analyze an array of samples on the sample plate 15. Therefore, in order to minimize human involvement, automation of the process is critical. The locations of the samples are typically pre-programmed into the computer that controls the MALDI-TOF apparatus so that during the analysis of the samples, the positioning mechanism 10 automatically repositions the sample plate 15 into the line of fire of the laser 5. Therefore, if any of the samples on the sample plate 15 are not properly positioned, the laser 5 is not likely to hit each of the samples. For instance, on the sample plate 15 depicted in FIG. 2, a 10xc3x9710 array of samples is positioned on the upper surface at spaced apart intervals. The positioning mechanism 10 moved into a target position with respect to centers of the desired location of each spot. The desired location of each spot assumes that center of each of the spots in the 10xc3x9710 array is constant.
Unfortunately, there are several shortcomings associated with the above described X-Y robotic apparatus (FIG. 3). Although the positioning mechanism 10 within the MALDI apparatus has positional accuracy with respect to movement of the sample plate 15, the X-Y robotic apparatus typically sold with a MALDI apparatus is not as precise with respect to accurate spotting or depositing of samples on the sample plate 15. Specifically, the spots in a 10xc3x9710 array of samples do not wind up being centered on the desired center targeted by the positioning mechanism 10. The array of 10xc3x9710 samples may have some samples that are accurately centered, and other samples that are off center by as much as half the width of the sample.
Part of the problem with such X-Y robotic apparatuses relates to the replaceable pipette tips 45 used to retrieve a sample and deposit the sample onto the sample plate 15. The pipette tips are not perfectly uniform in size and shape. Further, the tips are deformable and hence accurate positioning of samples is not possible. As well, the X-Y robotic apparatus may not have movement and location capabilities as precise as the movement and location capabilities of the positioning mechanism 10 of the MALDI apparatus.
Another problem relates to the pre-programmed settings for locating samples on sample plates 15 of such X-Y robotic apparatuses. Specifically, X-Y robotic apparatuses are not programmed to maximize the use of the space of the surface of the sample plate 15. For instance, each pair of adjacent samples in the 10xc3x9710 array of samples mentioned above is typically spaced apart by a distance greater than the diameter of the sample. There is a substantial amount of empty space on the surface of the sample plate 15 even with a 10xc3x9710 array of samples. Since the MALDI-TOF apparatus takes considerable time to analyze samples and requires human interaction to switch sample plates 15 in the mass spectrometer, it is advantageous to analyze as many samples as possible on a single sample plate. Current technology limits the number of samples that can be analyzed on a single sample plate 15.
There is a need for more precise positioning of samples on a sample plate and a need for maximizing the space on a sample plate in order to more efficiently utilize a mass spectrometry apparatus.
One object of the present invention is to provide a means for more precisely depositing samples on a sample plate of a mass spectrometry apparatus.
Another object of the present invention is to provide a means for maximizing the number of samples spotted on the surface of a sample plate of a mass spectrometry apparatus.
An aspect of the present invention relates to the use of an alignment plate to assist in accurate positioning of a pipette tip delivering samples onto a sample plate of a mass spectrometer.
Another aspect of the present invention relates to the use of an adaptor plate and an alignment plate with an X-Y manipulator device originally designed for liquid sample manipulation. Use of the adaptor plate and alignment plate of the present invention allows for new usage of the X-Y manipulator device. Specifically, the X-Y manipulator device can be used for spotting samples on a sample plate of a mass spectrometer with the alignment providing alignment holes for accurate positioning of samples on the sample plate.
In accordance with yet another aspect of the present invention, a plurality of alignment plates are used to guide a pipette tip toward a sample plate for accurate positioning of samples on the sample plate. A first alignment plate guides the pipette tip to spot an array of samples on the sample plate. After replacing the first alignment plate with a second alignment plate, a second array of samples can be used to guide the pipette tip to spot a second array of samples on the sample plate, where the second array of samples are located between, but offset from the first array of samples thereby maximizing the number of samples positionable on the sample plate.